DNA methylation profiling identifies global methylation differences and markers of adrenocortical tumors

Abstract

Context: It is not known whether there are any DNA methylation alterations in adrenocortical tumors.

Objective: The objective of the study was to determine the methylation profile of normal adrenal cortex and benign and malignant adrenocortical tumors.

Methods: Genome-wide methylation status of CpG regions were determined in normal (n = 19), benign (n = 48), primary malignant (n = 8), and metastatic malignant (n = 12) adrenocortical tissue samples. An integrated analysis of genome-wide methylation and mRNA expression in benign vs. malignant adrenocortical tissue samples was also performed.

Results: Methylation profiling revealed the following: 1) that methylation patterns were distinctly different and could distinguish normal, benign, primary malignant, and metastatic tissue samples; 2) that malignant samples have global hypomethylation; and 3) that the methylation of CpG regions are different in benign adrenocortical tumors by functional status. Normal compared with benign samples had the least amount of methylation differences, whereas normal compared with primary and metastatic adrenocortical carcinoma samples had the greatest variability in methylation (adjusted P ≤ 0.01). Of 215 down-regulated genes (≥2-fold, adjusted P ≤ 0.05) in malignant primary adrenocortical tumor samples, 52 of these genes were also hypermethylated.

Conclusions: Malignant adrenocortical tumors are globally hypomethylated as compared with normal and benign tumors. Methylation profile differences may accurately distinguish between primary benign and malignant adrenocortical tumors. Several differentially methylated sites are associated with genes known to be dysregulated in malignant adrenocortical tumors.

Fig. 1.

Unsupervised heirarchical cluster analysis of normal, benign, primary malignant, and metastatic tissue samples using ANOVA and an adjusted value of P ≤ 0.01. Primary ACC and metastatic tissue samples are globally hypomethylated. Red regions represent hypermethylation and blue regions are hypomethylation.

Fig. 2.

Differential methylation across all tissue comparisons. A, Normal compared with benign tissue samples has the least number of differences in methylation, and they are predominantly hypermethylated (104 total, 34 hypomethylated, and 70 hypermethylated). Primary and metastatic ACC samples compared with normal tissue samples have the greatest number of differences in methylation, and they are predominantly hypomethylated (primary ACC vs. normal, 24,229 total, 19,689 hypomethylated 4,540 hypermethylated, and metastatic vs. normal samples 21,736 total, 17,569 hypomethylated, and 4,167 hypermethylated). B, Primary and metastatic ACC samples compared with benign tissue samples have the next largest differences in methylation, and they are also predominantly hypomethylated (primary ACC vs. benign, 13,727 total, 8,824 hypomethylated, and 4,903 hypermethylated and metastatic vs. benign, 11,849 total, 7,650 hypomethylated, and 4,199 hypermethylated). Primary ACC samples compared with metastatic samples, however, have only 3799 differentially methylated sites, and they are predominantly hypermethylated (1556 hypomethylated and 2243 hypermethylated).

Fig. 3.

Principal component analysis of normal, benign, primary malignant, and metastatic tissue samples. Using ANOVA and an adjusted value of P ≤ 0.01, 44.5% of differentially methylated genes can separate the four tissue categories. Normal and benign tissue samples cluster more closely and have less variation across the individual samples. Primary malignant and metastatic tissue samples cluster separately from the normal and benign samples and each other and have more variability across each sample.

Fig. 4.

A, Percentage differences of CpG methylation probe location and functional genomic differences between normal, benign, primary, and metastatic malignant tissue samples. CpG locations in island, shore, shelf, and other. B, Methylation differences by functional genomics (promoter, body, 3′UTR, and intergenic regions). C, Methylation differences by RNA coding and noncoding regions. Normal and benign tissue samples have the least number of differentially methylated sites, but these sites are predominantly in the other/open sea and promoter regions of the genome as well as RNA coding regions.